The invention generally relates to vibrational microscopy and imaging systems, and relates in particular to vibrational imaging systems employing coherent Raman scattering.
Conventional vibrational imaging techniques include, for example, infrared microscopy, spontaneous Raman microscopy, and coherent anti-Stokes Raman scattering microscopy.
Infrared microscopy, which generally involves directly measuring the absorption of vibrational excited states in a sample, is limited by poor spatial resolution due to the long wavelength of infrared light, as well as by a low penetration depth due to a strong infrared light absorption by the water in biological samples.
Raman microscopy records the spontaneous inelastic Raman scattering upon a single (ultraviolet, visible or near infrared) continuous wave (CW) laser excitation. Raman microscopy has improved optical resolution and penetration depth as compared to infrared microscopy, but the sensitivity of Raman microscopy is rather poor because of the very low spontaneous Raman scattering efficiency; a Raman scattering cross section is typically on the order of 10−30 cm2. This results in long averaging times per image, which limits the biomedical applications of Raman microscopy.
Coherent anti-Stokes Raman (CARS) microscopy systems provide increased scattering signal from a sample due to coherent excitation. CARS microscopy systems use two pulsed excitation laser beams (pump and Stokes beams) with a frequency difference that matches the molecular vibration frequency of the chemical species to be imaged. As a result of interaction of the chemical species to be imaged with the difference frequency between the pump and Stokes beams, new illumination is generated at the sample at the anti-Stokes frequency, which is detected as the output signal in CARS microscopy. Imaging speeds up to video-rate have been achieved from highly resonant samples.
The CARS process, however, also excites a high level of background from the vibrationally non-resonant specimen. Such a non-resonant background not only distorts the CARS spectrum of the resonant signal from dilute samples but also carries the laser noise, significantly limiting the application of CARS microscopy on both spectroscopy and sensitivity perspectives. Various techniques have been developed to suppress this background, as disclosed, for example, in U.S. Pat. Nos. 6,798,507 and 6,809,814, but such systems each provides an anti-Stokes signal that is at least somewhat reduced by the background suppression.
Moreover, the specificity of the anti-Stokes signals for certain target species is limited because many chemical species may have a vibrational response at multiple frequencies. For example, FIG. 1 shows at 10 a Raman spectrum for the bioactive molecule adenosine triphosphate (ATP), the chemical formula for which is shown at 12 in FIG. 2. Note that because ATP has many different types of atomic bonds, it has several Raman active peaks that together provide a characteristic vibrational signature of the molecules.
The specificity is limited since many different chemical species (e.g., one target species and one non-target species) may have some of the same bonds (e.g., O—H) that provide the same vibrational response at the anti-Stokes frequency to the excitation illumination, making distinguishing between the two chemical species difficult or impossible based on a single anti-Stokes frequency.
Spectroscopy imaging systems have also been developed in which a broadband pulse is dispersed onto a multi-channel detector (photodiode-array or CCD) after passing through the focus, such that all spectral components can be individually detected. For example, synchronized broadband and narrowband pulse trains may be provided from mode-locked lasers. The combined pulse trains are provided to a laser-scanning microscope, and the nonlinear sample interaction occurs in the focus of the laser-scanning microscope. Output radiation is then provided to a dispersion device such as a grating or prism and then onto a multi-channel detector such as a photodiode array of a CCD after passing through the focus. Because of the use a spectrometer, images can only be achieved by slow stage scanning or low-throughput de-scanned detectors.
Such an approach is also difficult to unify with the high sensitivity detection schemes that require processing electronics such as a lock-in amplifier because every spectral component would need its own electronics. Furthermore spectroscopy is difficult to combine with laser-scanning microscopy because after passing through the sample the beam can move on the spectrometer and thus hinder the spectrum acquisition.
There is a need, therefore, for a microscopy imaging system that provides improved sensitivity and specificity. There is a further need for a microscopy imaging system that probes multiple Raman vibrations simultaneously to extract a spectral fingerprint that is free from spectral interference from other atomic bonds within a sample.